Project description:ATAC-seq was performed, mapped, and analyzed as previously described (PMID: 34446717: \\"ATAC-seq was performed on 50,000 cells per replicate as described in Buenrostro et al. (with modifications based on Corces et al.), on EpiSCs and PSM-differentiated cell populations at desired time-points. Libraries were generated using the Ad1_noMX and Ad2.1–2.16 barcoded primers64 and amplified for 10 total PCR cycles. Libraries were purified with AMPure XP beads to remove contaminating primer dimers and fragments >1,000 bp. Library quality was assessed using the Fragment analyzer and quantitated by Qubit assay. The libraries were sequenced with 50 bp paired-end reads on a Next-Seq 500 Sequencer (Illumina) at the DanStem Genomics Platform (University of Copenhagen, Copenhagen, Denmark).\\"). For all conditions, two biological replicate samples were collected from independent experiments. Library quality was assessed using the Fragment Analyzer and quantitated by Qubit assay. The libraries were sequenced with 50 bp paired-end reads on a NextSeq 500 Sequencer (Illumina) at the DanStem/reNEW Genomics Platform (University of Copenhagen, Copenhagen, Denmark). Prediction of cis-regulatory elements (CREs) and gene annotation was done using rGREAT (v4.0.4) [PMID: 20436461],[PMID: 36040971] or HOMER (v.4.7)[PMID: 20513432]
Project description:CD3+ T cells were enriched using the EasySep human T cell isolation kit (Stem cell technology). T cells from normal controls or patients with CARD9 mutations were co-cultured with monocytes of one normal control in the presence of heat killed candida. After 3 days, T cell were enriched again to remove the monocytes and total RNA was extracted from the T cells for the RNA-sequencing (RNASeq) evaluation. Targeted RNA sequencing library preparation was carried out using the Ion AmpliSeq Transcriptome Human Gene Expression Kit (Life Technologies), which profiles more than 20,000 human genes; each amplicon (~150 bp) represents a unique targeted gene (one transcript per gene). For library preparation, each sample was run in duplicate, and a cDNA library was generated from a minimum of 10 ng of total RNA. The cDNA was barcoded and amplified with Ion AmpliSeq technology, and the amplified cDNA Libraries were evaluated for quality and quantified with Agilent Bioanalyzer high-sensitivity chip. Libraries were then diluted to 100 pM and pooled equally, with 4 individual samples per pool. Pooled libraries were amplified and enriched with the Ion Chef System (Life Technologies). Templated libraries were then sequenced on an Ion Torrent Proton sequencing system (Life Technologies) with Ion PI HiQ kit and chip version 3. We performed gene-level differential expression analysis of targeted RNASeq data using R (v.3.5.3) and the Bioconductor packages DESeq2 (v.1.22.2).
2021-08-12 | GSE181718 | GEO
Project description:High-throughput surveys of SARS-related coronavirus RBDs using yeast display of barcoded libraries
Project description:Acute kidney injury (AKI) is an important contributor to the development of chronic kidney disease (CKD). We performed miRNA and mRNA sequencing on biobanked human kidney tissues obtained in the routine clinical care of patients with the diagnoses of AKI and minimal change disease (MCD), in addition to nephrectomized (Ref) tissue from individuals without known kidney disease. From all renal biopsy samples, 2 cryosections (10 μM) including the entire cross-section of the tissue were placed directly into PicoPure RNA extraction buffer. RNA was isolated using the PicoPure isolation kit. Total RNA was evaluated for quantity and quality using an Agilent Bioanalyzer. Approximately 10 ng of total RNA were used for each sample’s library preparation. Ribosomal RNA was removed using RiboGone – Mammalian Kit protocol. After rRNA depletion, double-stranded cDNA was synthesized and amplified using the SMARTer Universal Low Input RNA Kit protocol. The cDNA was sheared by Covaris, and the library was prepared and barcoded following the Ion Plus Fragment Library Kit protocol. Each resulting barcoded library was quantified and quality assessed by Agilent Bioanalyzer. Multiple libraries were pooled in equal molarity. Finally, 8 μL of 100 pM pooled libraries were loaded into lanes of an Illumina HiSeq 4000. At least 30M reads per library were generated.
Project description:Most of small RNA library construction methods are based on RNA ligases, which prefer to join the molecules (small RNAs and adapters) that can anneal to each other and form a ligase favoured structure. Different platforms for next generation sequencing use different adapter sequences, causing the cloning bias. Adapters with degenerated nucleotides at the ligating ends (High Definition, HD adapters) were developed to reduce the cloning bias. However, above 90% of the cloning products is adapter dimer when the current available commercial kits and their corresponding protocols are used. Here we adopted and further improved a method demonstrated in a publically available patent (http://www.google.com/patents/WO2011056866A2?cl=en). Using the improved method, we constructed the small RNA libraries by using the total RNA of chondrosarcoma cell line. The adapter dimer was significantly reduced. The small RNA sequences were also analysed. The small RNA libraries of cultured chondrosarcoma cell line were constructed by using an improved protocol where high-definition (HD) adapters were used.
Project description:Most of small RNA library construction methods are based on RNA ligases, which prefer to join the molecules (small RNAs and adapters) that can anneal to each other and form a ligase favoured structure. Different platforms for next generation sequencing use different adapter sequences, causing the cloning bias. Adapters with degenerated nucleotides at the ligating ends (High Definition, HD adapters) were developed to reduce the cloning bias. However, above 90% of the cloning products is adapter dimer when the current available commercial kits and their corresponding protocols are used. Here we adopted and further improved a method demonstrated in a publically available patent (http://www.google.com/patents/WO2011056866A2?cl=en). Using the improved method, we constructed the small RNA libraries by using the total RNA of Medicago truncatula leaf tissue. The adapter dimer was significantly reduced. The small RNA sequences were also analysed. The small RNA libraries of medicago truncatula leaves were constructed by using an improved protocol where high-definition (HD) adapters were used.
Project description:The submitted dataset contains raw files from 96 synthetic peptide libraries, using either HCD or ETD as fragmentation technique. The synthesized 96 tryptic peptide libraries containing >100,000 unmodified peptides plus their corresponding >100,000 phosphorylated counterparts with precisely known sequences and modification sites. All these libraries were subjected to LC-MS/MS on an Orbitrap mass spectrometer using HCD and ETD fragmentation. The generated mass spectrometric data deposited in this database can be used in numerous ways to develop, evaluate and improve experimental and computational proteomic strategies. Raw MS data files were converted into Mascot generic format files (MGF) using Mascot Distiller (2.4.2.0, www.matrixscience.com). Important parameters included: i) signal to noise ratio of 20 for MS/MS and ii) time domain off (no merging of spectra of the same precursor). The MGF files were searched against human IPI v3.72 including the sequences of all 96 libraries,using the Mascot search engine (2.3.1, 24). Search settings: Decoy search using a randomized version of the human IPI v3.72 including the sequences of all 96 libraries was enabled; monoisotopic peptide mass (considering up to two 13C isotopes); trypsin/P as protease; a maximum of four missed cleavages; peptide charge +2 and +3; peptide tol. +/- 5 ppm; MS/MS tol. +/- 0.02 Da; instrument type ESI-Trap (for HCD data) or ETD-Trap (for ETD data) respectively; variable modifications: oxidation (M), phospho (ST), phospho (Y). The result files were exported to pepXML and Mascot XML with default options provided by Mascot.
Project description:The goal of this study is to identify novel target genes of DVL3 in two breast cancer cell lines Methods:Total RNA was isolated using the Aurum™ Total RNA Mini Kit (Bio Rad) and library preparation and sequencing were performed at Center for Biotechnology & Genomics of Texas Tech University. RNA quality was determined using RNA Screen Tape (Agilent). Ribosomal RNA depletion was achieved using NEB Next rRNA Depletion Kit (Human/Mouse/Rat) (NEB # E6310X). RNA fragmentation, double stranded cDNA and adaptor ligation was generated using NEBNext Ultra II Directional RNA Library Prep according to the manufacturer’s protocol (NEB # E7760L). PCR enriched libraries were quantified by Qubit and equimolar indexed libraries (different samples had different indexes for multiplexing) were pooled. Pooled libraries were quantitatively checked using the Agilent Tapestation 2200 and quantified using Qubit. The libraries were then diluted to 200 pM and spiked with 2% phiX libraries (Illumina control). The transcriptome sequencing was performed on the barcoded stranded RNA-Seq libraries using Illumina NovaSeq 6000 SP flow cell, paired-end reads (2 × 50 bp).
Project description:Most of small RNA library construction methods are based on RNA ligases, which prefer to join the molecules (small RNAs and adapters) that can anneal to each other and form a ligase favoured structure. Different platforms for next generation sequencing use different adapter sequences, causing the cloning bias. Adapters with degenerated nucleotides at the ligating ends (High Definition, HD adapters) were developed to reduce the cloning bias. However, above 90% of the cloning products is adapter dimer when the current available commercial kits and their corresponding protocols are used. Here we adopted and further improved a method demonstrated in a publically available patent (http://www.google.com/patents/WO2011056866A2?cl=en). Using the improved method, we constructed the small RNA libraries by using the total RNA of Medicago truncatula leaf tissue. The adapter dimer was significantly reduced. The small RNA sequences were also analysed.